Part:BBa_K4595021

Prbcl-gshA-gshB-Tpsbc

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 145
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 145
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 145
  Illegal BamHI site found at 1210
  Illegal BamHI site found at 1683
  Illegal BamHI site found at 2187
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 145
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 145
  Illegal NgoMIV site found at 2164
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

It is a complex component composed of the promoter Prbcl, target genes gshA, gshB and terminator Tpsbc, which is responsible for the high expression of Gsh synthesis and thus the intracellular Gsh surplus of the engineered bacteria to withstand the extreme environment that may be caused by the increasing climate crisis.

Fig.1 The glutathione metabolism.

The abbreviations are as follows GSH, glutathione (reduced form); GSSG, glutathione disulfide (oxidized glutathione); GshA, c-glutamylcysteine ligase; GshB, glutathione synthase; Ggt, c-glutamyltranspeptidase; GR, glutathione reductase; AA, amino-acid.

Prbcl

Pcpc560 is a super-strong promoter containing two predictive promoters from the cpcB gene and 14 predictive transcription factor binding sites (TFBSs). This efficient promoter enables heterologous proteins to be expressed robustly in Synechocystis PCC6803.

gshA and gshB

Glutathione (GSH) is an important antioxidant containing sulfur compounds. It is a tripeptide composed of three amino acids (cysteine, glycine, and glutamic acid) and non-protein thiols. GSH is usually in a reduced state and plays a critical role in detoxifying reactive oxygen species (ROS) and free radicals that cause oxidative stress. ROS can change the spatial structure of proteins by obtaining electrons, leading to the degeneration of many protein-dependent chemical substances and even DNA, further affecting the normal life activities of the cell. However, the reduced form of GSH can protect the chemical structure of proteins by providing extra electrons for ROS and free radicals. GSH peroxidase catalyzes this process. According to the reference, we founded that Arthrospira sp. PCC 8005 activates the cell's Gsh synthesis pathway to resist radiation hazards such as oxde-induced protein denaturation and DNA damage rather than inducing classical antioxidant or DNA repair systems like superoxide dismutase (SOD) enzymes and RecA proteins. To enhance our engineered bacteria's resistance to extreme environments, we overexpressed GSH synthesis in advance in Synechocystis PCC6803.This measure was aimed at equipping the bacterium with the capacity to withstand potential threats arising from the escalating climate crisis. By enhancing its adaptation to extreme environments, this modified strain may not only survive but also potentially mitigate any adverse effects. Moreover, it has the potential to offer insights into the reclamation of dangerous land areas and the exploration of space.There is a natural Gsh synthesis pathway in Synechocystis PCC6803 mediated by two ATPdependent reactions which catalyzed by γ-glutamylcysteine synthelase (γ-GCs) and glutathione synthase (GS). γ-Gcs and GS are encoded by two different genes, the gshA and gshB genes, respectively. The gshA gene sequence encodes γ-glutamylcysteine synthelase (gamma-GCs), which binds glutamate and cysteine to form γ-glutamylcysteine metabolites. gshB is the gene sequence encoding glutamine synthetase (GS), which generates glutathione by adding glycine to γ-glutamylcysteine metabolites (Fahey, 2013).

Molecular cloning

In order to construct the desired plasmids, we employed the E.coli TOP10 amplification method. Firstly, we performed PCR amplification using specific primers for each plasmid, which results in the generation of linearized fragments harboring the target sequences in a high copy number. These fragments were then connected into complete plasmids using restriction enzyme digestion and enzyme ligation procedures. After transfer to E.coli TOP10, colony PCR was used to confirm successful construction of the plasmid. Subsequently, the plasmids were further amplified to obtain sufficient quantities for further experiments. Finally, the complete plasmids were introduced into Synechocystis PCC6803 cells and their successful integration was verified through colony PCR analysis.

Fig.2 Colony PCR result of Prbcl-dsup-Tpsbc-Prbcl-gshA-gshB-Tpsbc transformed E.coli TOP 10

The band of Prbcl-dsup-Tpsbc-Prbcl-gshA-gshB-Tpsbc from colony PCR is about 4700bp, identical to the theoretical length of 4746bp estimated by the designed primer location which could demonstrate that this target plasmid had successfully transformed into E.coli TOP 10.